Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host cells.
Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host cells.
复制标题
使用荧光诱导和蔗糖反选择来鉴定宿主细胞内表达的结核分枝杆菌基因。
作者：
J. Triccas;F. Berthet;V. Pelicic;Brigitte Gicquel
鉴定结核分枝杆菌在宿主细胞内表达的基因将大大有助于开发新的策略来对抗结核病。通过将维多利亚水母绿色荧光蛋白（GFP）的天然荧光与枯草芽孢杆菌SacB蛋白的反选择特性相结合，M.已经分离出显示增强的体内活性的结核病启动子。用含有M.控制gfp和sacB表达的结核启动子，以及通过荧光激活细胞分选回收的荧光细菌。sacB的表达用于消除巨噬细胞外具有强启动子活性的克隆，从而分离出7个含有M.结核病启动子具有更大的细胞内活性。所鉴定的基因产物显示出与来自其他生物的蛋白质的相似性，其功能包括营养利用、保护免受氧化应激和防御外源性物质。这些提出的功能与宿主细胞内遇到的条件一致，因此表明分离的启动子/基因的增强活性可能代表M.结核病，以提高细胞内存活和促进感染。
The identification of Mycobacterium tuberculosis genes expressed within host cells would contribute greatly to the development of new strategies to combat tuberculosis. By combining the natural fluorescence of the Aequoria victoria green fluorescent protein (GFP) with the counterselectable property of the Bacillus subtilis SacB protein, M. tuberculosis promoters displaying enhanced in vivo activity have been isolated. Macrophages were infected with recombinant Mycobacterium bovis bacille Calmette-Guérin containing a library of M. tuberculosis promoters controlling gfp and sacB expression, and fluorescent bacteria recovered by fluorescence-activated cell sorting. The expression of sacB was used to eliminate clones with strong promoter activity outside the macrophage, resulting in the isolation of seven clones containing M. tuberculosis promoters with greater activity intracellularly. The gene products identified displayed similarity to proteins from other organisms whose functions include nutrient utilization, protection from oxidative stress and defence against xenobiotics. These proposed functions are consistent with conditions encountered within the host cell and thus suggest that the augmented activity of the isolated promoters/genes may represent strategies employed by M. tuberculosis to enhance intracellular survival and promote infection.
DOI：
10.1073/pnas.94.20.10955
发表时间：
1997-09-30
影响因子：
11.1
作者：
Pelicic, V;Jackson, M;Guilhot, C
通讯作者：
Guilhot, C
影响因子：
15.9
作者：
Clemens, DL
通讯作者：
Clemens, DL
影响因子：
56.9
作者：
Valdivia, RH;Falkow, S
通讯作者：
Falkow, S